Journal of Medicine and Medical Sciences Vol. 6(7) pp. 156-161, September, 2015
DOI: http:/dx.doi.org/10.14303/jmms.2012.177
Available online http://www.interesjournals.org/JMMS
Copyright © 2015 International Research Journals
Full Length Research Paper
Hepatoprotective potentials of the methanolic leaf extract of
Jatropha curcas (LINN) on cadmium induced toxicity in rabbit
Adejumobi A.E.*, Areola J.O and Babalola O.O.
Department of Biochemistry, Obafemi Awolowo University, Ile Ife, Nigeria
*Corresponding Author E- mail: doctorbablo@yahoo.com
Abstract
This study was carried out to investigate the possible hepatoprotective potentials of methanolic leaf
extract of Jatropha curcas Linn on cadmium induced hepatotoxicity and oxidative stress in rabbits.
Sixteen matured rabbits were used for the study; they were divided into four groups and treated as
follows: Group I (control) was treated with normal saline (0.85% w/v NaCl) at 2 ml/kg body weight per day.
Group II were treated with only cadmium chloride at 1.25 mg/kg body weight per day. Groups III and IV
were pre-treated with 300 mg/kg body weight per day of Jatropha curcas extract and vitamin E
respectively, one hour later, cadmium chloride was administered to them at 1.25 mg/kg body weight. All
the animals were treated orally for 10 days. The animals were sacrificed on day 11, blood, liver and
kidneys were removed and preserved for biochemical analyses. The liver samples were digested with
mixture of concentrated trioxonitrate (V) acid (HNO3) and hydrochloric acid (HCl) (1:4 v/v). Cadmium
concentrations in the liver were determined by atomic absorption spectroscopy (AAS). The concentrations
of total plasma protein, albumin, uric acid and the activities of hepatic marker enzymes; alanine
aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyltransferase (GGT) were
determined in the plasma and liver homogenates by standard procedures. The result of phytochemical
screening revealed that flavonoids, tannins, terpenoids steroids, phlobatanins and saponins were present
in the extract. The analysis by atomic absorption spectroscopy of the liver revealed that there were traces
of cadmium (0.01 μg/g) in the liver of animals pre-treated with vitamin E and Jatropha curcas extract while
the concentration of cadmium in the liver of only cadmium group was 0.14 µg/g. Additionally, the activities
of hepatic marker enzymes (AST, ALT, GGT) were elevated in cadmium only treated group but pretreatment with methanolic extract of Jatropha curcas reduced significantly (p<0.05) the activities of these
enzymes. The total protein and uric acid concentrations were also significantly reduced in cadmium
treated group when compared with the extract pre-treated group. Comparison of the activities of hepatic
marker enzymes (ALT, AST and GGT) in vitamin E and Jatropha curcas extract pre-treated groups revealed
that ALT, AST and GGT activities were not significantly different in these groups. Also, albumin and uric
acid concentrations were not significantly different between the groups. The results of biochemical
studies of blood samples and liver homogenates revealed significant increase in the levels of maker
enzymes reflecting liver injury caused by cadmium while pre-treatment with Jatropha curcas extract
reduced the activities of the enzymes. Oral administration of methanolic extract of Jatropha curcas at 300
mg/kg body weight in this study was able to reduce the hepatotoxic effects of cadmium toxicity at 1.25
mg/kg body weight. This suggests that methanolic leaf extract of Jatropha curcas might possess
hepatoprotective effect. The results of this investigation has further confirmed the inherent therapeutic
properties of Jatropha curcas and also justified its uses in traditional medicine to cure various ailments in
Asia, Africa and Latin America.
Keywords: Jatropha curcas, Cadmium, Rabbit, Heavy Metals, Hepatotoxicity.
INTRODUCTION
Heavy metals occur as natural constituents in the earth
crust and are persistent environmental contaminants
since they are not degraded or destroyed. To a large
extent, they enter the body system through food, air, and
water and bio-accumulate over a period of time.
Cadmium is a classic- elemental toxicant that shows
Adejumobi et al. 157
various adverse effects and can be fatal depending on
the dose. The frequency of exposure to cadmium is
gradually on the rise as a result of increase in the
production and usage of materials like tobacco, cement,
phosphate fertilizer and other materials containing
cadmium. Cadmium is a non-essential toxic heavy metal,
which is known to affect cellular processes through
membrane damage, disruption of electron transport,
enzyme inhibition and DNA alteration (Godt et al., 2006,
Babalola and Areola, 2010).
Upon acute exposure to cadmium, hepatotoxicity is
indicated by changes such as swelling of hepatocytes,
fatty changes, and focal necrosis or necrosis in a wide
area. Cadmium accelerates lipid peroxidation by
stimulating the peroxidation chain reaction in the target
organs, resulting in the generation of reactive oxygen
species (Oteiza et al., 1999).
Liver is one of the largest organs in human body and
the chief site for biotransformation of toxic materials. So it
plays an important role in the maintenance and regulation
of the body homeostasis (Ward and Daly, 1999).
However if the liver is continuously exposed to
environmental toxins which include the heavy metals,
various ailments like liver cirrhosis and fatty liver disease
could result ( Subramonium and Pushpangadan, 1999).
There are not many drugs for the treatment of liver
damage, therefore, many folk remedies from plants origin
are tested for their potential antioxidant and
hepatoprotective effects in experimental animal model.
The demand for natural antioxidants has increased as
they are viewed as promising free radical scavengers.
With the current resurgence of scientific research in
herbal medicine, it is becoming clearer that the medicinal
herbs are becoming better alternatives to synthetic ones;
though many synthetic drugs in use today were derived
from medicinal herbs. According to one estimate, only
20% of the plant flora has been studied and 60% of
synthetic medicines owe their origin to plants (Cowan,
1999).
Jatropha curcas Linn is a multipurpose and drought
resistant perennial plant which belongs to the family
Euphorbiaceae. It is known commonly as Physic nut or
Purging nut, the oil produced from the nut of this plant is
used as a biodiesel. The methanolic leaf extract was
reported to have antiviral activity (Gupta et al; 1996). The
ethanolic extract of the defatted leaves and twigs had
been shown to have anti-leukemic activity (Hufford and
Oguntimehin, 1978). The butanolic extract of dried leaves
of Jatropha curcas and decoction of dried root bark was
shown to possess antispasmodic activity (Kambu et al.,
1990).
The current study on Jatropha curcas was designed to
investigate the possible hepatoprotective activity of its
leaf extracts on cadmium induced toxicity in rabbits.
MATERIALS AND METHODS
Plant Material
Fresh leaves of Jatropha curcas were collected in the
month of November, 2009 from Ikire town in Osun State,
Nigeria. They were identified and authenticated by Mr.
Ibhanesebor G.A. of the Department of Botany, O.A.U
Ife-Ife.
Reagents and Chemicals
All the reagents used in the study were of analytical
grade. They were obtained from British Drug House
Limited (BDH) London, Sigma Fine Chemicals Limited,
Upsalla, Sweden, Fluka Chemical Company Plc.
Germany. Reagent kits to assay for the enzymes were
obtained from Randox Laboratories Ltd, United Kingdom.
All solutions, buffers and reagents were prepared with
glass-distilled water.
Extract Preparation
The extract was prepared according to the procedure
described by Uche and Aprioku,( 2008). The leaves were
air dried for sixteen days in the laboratory after which
they were ground into fine powder. The powdered leaves
(500 g) were subjected to Soxhlet extraction using
methanol as solvent for 12 hours; the filtrate was
evaporated to dryness using Edward vacuum evaporator.
The yield was calculated to be 80.15g representing
16.03% .It was kept in air tight container for future
analyses.
Phytochemical Screening
The extract was screened for terpenoid, steroid, tannin,
saponin, flavonoid, cardiac glycoside and phlobatannin
according to standard procedures (Harborne 1998,
Edeoga et al., 2005)
Experimental Design
Sixteen female rabbits were used for this study. The
animals were allowed to acclimatize under laboratory
condition for two months. At the end of the
acclimatization, the animals were grouped randomly into
four (4 animals per group). Each group was treated orally
once per day for 10 days as follows: Group I (Control):
Animals in this group received 2 ml/kg body weight of
158 J. Med. Med. Sci.
normal saline. Group II (Cadmium only treated): Animals
in this group received 1.25 mg/kg body weight of
cadmium chloride only. Group III( Extract pre-treated):
Animals in this group were pre-treated with 300 mg/kg of
Jatropha curcas leaf extract and one hour later, cadmium
chloride was administered ( 1.25 mg/kg body weight).
Group IV (Vitamin E pre-treated): Animals in this group
were given vitamin E (300mg/kg body weight) before the
administration of cadmium chloride (1.25 mg/kg body
weight) after 1 hour. Prior to the administration of the
extract, it was reconstituted in olive oil at a concentration
equivalent to 300 mg/kg body weight. The animals were
sacrificed at the end of the treatment period, blood was
collected through cardiac puncture into clean bottles
containing anticoagulant, the liver and the kidneys were
excised for various biochemical analyses.
Assay of Enzyme Activities
Digestion of Liver Samples for AAS Analysis
RESULTS
The acid digestion of the liver was carried out as
described by Babalola and Areola (2010). The liver (1 g)
of each animal was homogenized in 5 ml of mixture of
concentrated HNO3 and HCl (1:4 v/v). The homogenates
were transferred into test tubes, covered with cotton wool
and left on bench overnight to solubilize the tissue. The
mixture was heated at 100oC in water bath for 20 minutes
and allowed to cool, followed by the addition of 1.0 ml of
hydrogen peroxide. The sample was then diluted to a
final volume of 25 ml with distilled water and stored in a
30ml polyethylene bottle for analyses by Atomic
Absorption Spectrophotometer.
The phytochemical tests revealed the presence of
flavonoids, steroids, terpenoids, tannins, phlobatannins,
and saponins while cardiac glycoside was absent as
shown in Table 1
The analysis by atomic absorption spectroscopy of the
liver revealed that there were traces of cadmium (0.01
μg/g) in the liver of animals pre-treated with vitamin E and
Jatropha curcas extract while the concentration of
cadmium in the liver of only cadmium group was 0.14
µg/g. The results of biochemical analyses of the plasma
from different treatment groups are shown in tables 2 and
3. Effects of Cadmium and Jatropha curcas on total
protein, albumin and globulin are displayed in Table 2
while Table 3 showed the effects on the hepatic marker
enzymes; ALT (Alanine aminotransferase), AST
(Aspartate alanine aminotransferase), GGT (Gamma
glutammyltransferase). Concentration of metabolites and
the activities of the enzymes were affected differently by
cadmium in treated groups when compared with the
control groups. It was evident from Table 3, where the
biochemical parameters were compared that Jatropha
curcas extract and vitamin E displayed similar effects on
the animals.
Preparation of Liver Homogenates and Blood Plasma
Liver samples (2g) were homogenized in 10 ml of cold
phosphate buffer (pH 7.4, 0.1 M) and centrifuged at 4000
rpm for 30 minutes. The supernatants were collected into
clean sterile bottles and kept in freezer for further
analyses. The blood collected from each animal in
heparinized bottle was centrifuged at 3000 rpm for 30
minutes. The supernatants were collected into clean
sterile bottles and kept in the freezer for further analyses.
The activities of aminotransferases (AST and ALT) were
assayed as described by Reitman and Frankel (1957)
using randox diagnostic kits. The activity of gamma
glutamyltransferase (GGT) was assayed according to the
method of Teitz (1957)) using randox diagnostic kits.
Statistical Analyses
Values of biochemical parameters were expressed as
Mean ± SEM. The statistical analysis was carried out
using analysis of variance (ANOVA) and Student t test.
Comparisons were made between different Groups,
values < 0.05 were considered as significant
DISCUSSION
Biochemical Estimation
Metabolites
The total protein concentration was determined using
Biuret reaction method as described by Cannon et al.,
(1974). Albumin concentration was determined using a
method as described by Pinnel and Northan (1978). Uric
acid in the liver and plasma was estimated as described
by Fossati et al., (1980).
Presence of secondary metabolites commonly referred to
as phytochemicals have been shown to be responsible
for various pharmacological properties of medicinal plants
(Okajuku et al., 2006). Presence of steroid, terpenoid,
tannin, flavonoid and saponin in the methanolic extract of
the leaf of Jatropha curcas agreed with previous findings
by Igbinosa, et al, 2009. Previous research work on
different parts of Jatropha curcas has revealed the
therapeutic potentials of the plants extracts as antimicro-
Adejumobi et al. 159
Table 1. Summary of Phytochemical Constituents of Methanolic
Leaf Extract of Jatropha curcas
Phytochemicals
Inference
Flavonoids
Steroids
Terpenoids
Tannins
Phlobatannins
Cardiac glycosides
Saponins
+ Present,
+
+
+
+
+
+
- Absent
Table 2. Effects of the extract of Jatropha curcas and Cadmium on concentration of Total protein,
Albumin and Globulin in the Plasma of treated Animals
Treatment group
Control
Cadmium
J. curcas + cadmium
Vit. E + cadmium
Total protein (mg/dl)
27.13 ± 3.84
10.35 ± 2.42
19.14 ± 1.90
17.76 ± 1.90
Albumin (mg/dl)
5.18 ± 2.34
2.69 ± 0.13
3.11 ± 0.13
3.28 ±0.72
Globulin (mg/dl)
21.95 ± 6.18
7.66 ± 2.55
16.03 ± 2.03
14.48 ± 1.18
The values are expressed as mean ± SEM.
Table 3. Effects of Jatropha curcas extract and Cadmium on AST, ALT and GGT Activities
in the Plasma
Treatment group
Control
Cadmium
J. curcas + cadmium
Vit. E + cadmium
AST (U/L)
ALT (U/L)
GGT (U/L)
38.00 ± 6.00
92.00 ± 8.00
41.00 ± 1.00
43.00 ± 7.00
16.40 ± 4.91
40.71 ± 4.08
11.76 ± 0.87
9.57 ±1.92
1.74 ± 0.58
31.27 ± 1.16
1.80 ± 0.55
1.74 ± 0.58
The values are expressed as mean ± SEM.
Table 4. Comparisons of the activities of Plasma AST, ALT, GGT, Total Protein, Uric Acid and Albumin
between Jatropha curcas Extract and Vitamin E Pre-treated Groups
PARAMETERS
ALT
AST
GGT
TOTAL PROTEIN
URIC ACID
ALBUMIN
Control
J.curcas + Cd
Vitamin E + Cd
P<0.05)
I6.40 ± 4.91
38.00 ± 6.00
1.74 ± 0.58
27.13 ± 3.84
0.21 ± 0.16
5.18 ± 2.34
11.76 ± 0.87
41.00 ±1.00
1.80 ± 0.55
19.14 ± 1.90
0.11 ± 0.01
3.106 ± 0.13
9.57 ± 1.92
43.00 ± 7.00
1.74 ± 0.58
17.76 ± 1.90
0.18 ± 0.07
3.28 ± 0.72
NS
NS
NS
S
NS
NS
Values are expressed as Mean ± SEM
bial (Ayelaagbe et al., 2000, Ayelaagbe, 2001 and
Igbinosa et al., 2009) and antiparasitic (Fagbenro
Beyioku, 1998). Phenolic compounds are known for their
antioxidant properties, especially flavonoids. Presence of
these antioxidant compounds in plants extracts is
responsible for their protective effects against various
degenerative diseases. They are able to protect the
tissues and organs because of their ability to scavenge
free radicals and prevent generation of other dangerous
reactive oxygen species (Sachin et al., 2010).
Protein synthesis is one of the major functions of the
liver. Thus total protein level in the plasma is often used
as an indicator for health status. The results of plasma
protein concentrations in different animal groups were
160 J. Med. Med. Sci.
presented in Table 2. Protein concentration reduced
significantly from 27.13 ± 3.84 g/dl in control to 10.35 ±
2.42 g/dl in cadmium only group. This reduction can be
attributed to destruction of hepatocytes by cadmium
(Bondy et al., 2000). Total protein concentrations were
higher in Jatropha curcas extract and vitamin E pretreated groups but lower in the cadmium only group.
These protective effects of Jatropha curcas and vitamin E
may be due to their antioxidant properties .The same
trend was observed in the plasma albumin
concentrations. Plasma albumin concentrations reduced
from 15.18 ± 2.34 g/dl in control to 2.69 ± 0.13 g/dl in
cadmium as shown in Table 2. The significant reduction
observed in the plasma albumin concentration may be an
indication of liver injury, but albumin synthesis is also
sensitive to amino acid supply thus nutrition state plays
important roles in albumin concentration (Harper, 1961).
High plasma activities of aspartate aminotransferase
(AST) and alanine aminotransferase (ALT) are
associated with inflammation or injury to liver cells, a
condition known as hepatocellular liver injury. Damage to
the liver typically results in a leak of these enzymes into
the bloodstream (Mumoli, et al., 2006). Both AST and
ALT are enzymes found in the hepatocytes, upon
destruction of hepatocytes or due to an increase in the
permeability of the hepatocyte membrane, these
enzymes are released to the blood and the levels are
consequently elevated (Blasco and Puppo, 1999).
Cadmium is an established toxic metal; the toxic effects
of cadmium manifest through the promotion of oxidative
stress in the tissues and organs where it bioaccumulates. Activities of these hepatic marker enzymes
were determined in the plasma of the experimental
animals. The increase in activity of alanine
aminotransferase in the plasma of animals in cadmium
only treated group from 16.40 ± 4.91 U/L in control to
40.71 ± 4.08 U/L confirmed the finding of Bondy et al.,
(2000), that cadmium causes peroxidative effect on the
plasma membrane of hepatocytes leading to enzyme
leakage into the extracellular fluid. Significant reduction in
the alanine aminotransferase activities were observed in
the Jatropha curcas extract and vitamin E treated groups
when compared with the control group. The activities
reduced from 16.40 ± 4.91 U/L to 11.76 ± 0.87 U/L and
9.57 ± 1.92 U/L in Jatropha curcas extract and vitamin E
pre-treated groups respectively. Vitamin E and Jatropha
curcas extract have antioxidant properties; they were
expected to ameliorate the toxic effects of cadmium in the
animals. The probable reason for this may be partial
inhibition of the enzymes synthesis in the presence of
cadmium.
Gamma glutamyltransferase (GGT) is another enzyme
used in clinical diagnosis of liver disorders. GGT levels
are elevated in disease of the bile ducts and in some liver
diseases (Mumoli, et al., 2006). Elevation of the enzyme
activities from 1.74 ± 0.58 U/L in control group to 31.27 ±
1.16 U/L in the cadmium only treated group also con-
firmed liver damage. This result is consistent with the
other results already discussed. Administration of
Jatropha curcas extract significantly reduced the activity
of the enzyme in the plasma significantly. Similar values
were recorded for the control and vitamin E group
showing complete protection against cadmium toxicity.
In conclusion, the results of this investigation has
further confirmed the inherent therapeutic properties of
Jatropha curcas and also justified its uses in traditional
medicine to cure various ailments in Asia, Africa and
Latin America (Burkill, 1994). Methanolic extract of the
leaf of Jatropha curcas can serve as a base for the
discovery of cheap and effective hepatoprotective drug.
Further research is required to investigate and isolate the
phytochemical(s) responsible for this activity.
ACKNOWLEDGEMENT
We are grateful to the staff of Drug Research and
Production Unit (DRPU), Faculty of Pharmacy, Obafemi
Awolowo University Ile-Ife, for the use of their Soxhlet
apparatus and vacuum pump evaporator.
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